Carbamate-derivatized nucleosides and oligonucleosides

ABSTRACT

Nucleosides and oligonucleosides functionalized to include carbamate functionality, and derivatives thereof. In certain embodiments, the compounds of the invention further include steroids, reporter molecules, reporter enzymes, lipophilic molecules, peptides or proteins attached to the nucleosides through the carbamate group.

This application is a division of U.S. patent application Ser. No.09/688,394 (now U.S. Pat. No. 6,322,987), filed Oct. 16, 2000, which isa division of U.S. patent application Ser. No. 09/372,856 (now U.S. Pat.No. 6,166,188), filed Aug. 12, 1999, which is a division of U.S. patentapplication Ser. No. 08/713,742 (now U.S. Pat. No. 6,111,085), filedSep. 13, 1996.

FIELD OF THE INVENTION

This application is directed to nucleosides, oligonucleotides andoligonucleosides that are functionalized with carbamate moieties. Thecarbamate moieties are used for linking various conjugate groups to thenucleosides, oligonucleotides or oligonucleosides. Suitable conjugategroups include, but are not limited to, steroids, reporter molecules,reporter enzymes, lipophilic molecules, cleaver molecules, peptides andproteins.

BACKGROUND OF THE INVENTION

Messenger RNA (mRNA) directs protein synthesis. Antisense methodology isthe complementary hybridization of relatively short oligonucleotides tomRNA or DNA such that the normal, essential functions of theseintracellular nucleic acids are disrupted. Hybridization is thesequence-specific hydrogen bonding via Watson-Crick base pairs ofoligonucleotides to RNA or single-stranded DNA. Such base pairs are saidto be complementary to one another.

The naturally occurring events that provide the disruption of thenucleic acid function, discussed by Cohen in Oligonucleotides: AntisenseInhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989)are thought to be of two types. The first, hybridization arrest, denotesthe terminating event in which the oligonucleotide inhibitor binds tothe target nucleic acid and thus prevents, by simple steric hindrance,the binding of essential proteins, most often ribosomes, to the nucleicacid. Methyl phosphonate oligonucleotides (Miller, et al., Anti-CancerDrug Design 1987, 2, 117) and α-anomer oligonucleotides are the two mostextensively studied antisense agents which are thought to disruptnucleic acid function by hybridization arrest.

The second type of terminating event for antisense oligonucleotidesinvolves the enzymatic cleavage of the targeted RNA by intracellularRNase H. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotideanalog hybridizes with the targeted RNA and this duplex activates theRNase H enzyme to cleave the RNA strand, thus destroying the normalfunction of the RNA. Phosphorothioate oligonucleotides are the mostprominent example of an antisense agent that operates by this type ofantisense terminating event.

Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs as antisense agents fordiagnostics, research reagents and therapeutic compounds. As researchreagents oligonucleotides and oligonucleotide analogs find various usesincluding, but not limited to, probes and primers. For diagnostics,oligonucleotides and oligonucleotide analogs can be used in cell freesystems, in vitro, ex vivo or in vivo. Currently a number ofoligonucleotide based drugs are being tested in human clinical trialsfor various disease states including AIDS, against various cancers andfor various systemic disease resulting from inappropriate immuneresponses. The antisense oligonucleotides and oligonucleotide analogscan be functionalized with various conjugate groups to modify certain oftheir properties. Thus reporter groups can be conjugated to theoligonucleotides or oligonucleotide analogs to assist in identificationand location of the compounds in various testing medium includingreagents, cellular products or digests, cell systems and organisms.Other conjugate groups can be utilized for transport, binding and uptakemodulation, modification of solubility characteristics, analyticalinstrument identification and response and other useful properties knownin the art.

Ramirez, et al., J. Am. Chem. Soc. 1982, 104, 5483, introduced thephospholipid group 5′-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl)into the dimer TpT independently at the 3′ and 5′ positions.Subsequently Shea, et al., Nuc. Acids Res. 1990, 18, 3777, disclosedoligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linkedto a 5′-phosphate on the 5′-terminus of the oligonucleotide. Certain ofthe Shea, et. al. authors also disclosed these and other compounds inpatent application PCT/US90/01002. A further glucosyl phospholipid wasdisclosed by Guerra, et al., Tetrahedron Letters 1987, 28, 3581.

In other work, a cholesteryl group was attached to the inter-nucleotidelinkage between the first and second nucleotides (from the 3′ terminus)of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013and further by Letsinger, et al., Proc. Natl. Acad. Sci. USA 1989, 86,6553. The aromatic intercalating agent anthraquinone was attached to the2′ position of a sugar fragment of an oligonucleotide as reported byYamana, et al., Bioconjugate Chem. 1990, 1, 319. The same researchersplaced pyrene-1-methyl at the 2′ position of a sugar (Yamana et. al.,Tetrahedron Lett. 1991, 32, 6347).

Lemairte, et al., Proc. Natl. Acad. Sci. USA 1986, 84, 648; andLeonetti, et al., Bioconjugate Chem. 1990, 1, 149. The 3′ terminus ofthe oligonucleotides each include a 3′-terminal ribose sugar moiety. Thepoly(L-lysine) was linked to the oligonucleotide via periodate oxidationof this terminal ribose followed by reduction and coupling through aN-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates aredescribed in European Patent application 87109348.0. In this instancethe lysine residue was coupled to a 5′ or 3′ phosphate of the 5′ or 3′terminal nucleotide of the oligonucleotide. A disulfide linkage has alsobeen utilized at the 3′ terminus of an oligonucleotide to link a peptideto the oligonucleotide as is described by Corey, et al., Science 1987,238, 1401; Zuckermann, et al., J. Am. Chem. Soc. 1988, 110, 1614; andCorey, et al., J. Am. Chem. Soc 1989, 111, 8524.

Nelson, et al., Nuc. Acids Res. 1989, 17, 7187 describe a linkingreagent for attaching biotin to the 3′-terminus of an oligonucleotide.This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commerciallyavailable from Clontech Laboratories (Palo Alto, Calif.) under the name3′-Amine on. It is also commercially available under the name3′-Amino-Modifier reagent from Glen Research Corporation (Sterling,Va.). This reagent was also utilized to link a peptide to anoligonucleotide as reported by Judy, et al., Tetrahedron Letters 1991,32, 879. A similar commercial reagent (actually a series of such linkershaving various lengths of polymethylene connectors) for linking to the5′-terminus of an oligonucleotide is 5′-Amino-Modifier C6. Thesereagents are available from Glen Research Corporation (Sterling, Va.).These compounds or similar ones were utilized by Krieg, et al.,Antisense Research and Development 1991, 1, 161 to link fluorescein tothe 5′-terminus of an oligonucleotide. Other compounds of interest havealso been linked to the 3′-terminus of an oligonucleotide. Asseline, etal., Proc. Natl. Acad. Sci. USA 1984, 81, 3297 described linkingacridine on the 3′-terminal phosphate group of an poly (Tp)oligonucleotide via a polymethylene linkage. Haralambidis, et al.,Tetrahedron Letters 1987, 28, 5199 report building a peptide on a solidstate support and then linking an oligonucleotide to that peptide viathe 3′ hydroxyl group of the 3′ terminal nucleotide of theoligonucleotide. Chollet, Nucleosides & Nucleotides 1990, 9, 957attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the5′ terminal phosphate of an oligonucleotide. They then used thebifunctional linking group SMPB (Pierce Chemical Co., Rockford, Ill.) tolink an interleukin protein to the oligonucleotide.

An EDTA iron complex has been linked to the 5 position of a pyrimidinenucleoside as reported by Dreyer, et al., Proc. Natl. Acad. Sci. USA1985, 82, 968. Fluorescein has been linked to an oligonucleotide in thesame manner as reported by Haralambidis, et al., Nucleic Acid Research1987, 15, 4857 and biotin in the same manner as described in PCTapplication PCT/US/02198. Fluorescein, biotin and pyrene were alsolinked in the same manner as reported by Telser, et al., J. Am. Chem.Soc. 1989, 111, 6966. A commercial reagent, Amino-Modifier-dT, from GlenResearch Corporation (Sterling, Va.) can be utilized to introducepyrimidine nucleotides bearing similar linking groups intooligonucleotides.

Carbamate linkages have been utilized to link conjugate groups tooligonucleotides at the 5′ position as reported by DeVos, et al.,Nucleosides Nucleotides, 9, 259, 1990, Wachter, et al., Nucleic AcidsRes., 14, 7985, 1986, and Gottikh, et. al., Tetrahedron Lett., 31, 6657,1990. Carbamate linkages have not been used for link conjugate groups tothe 2′ nor the 3′ position of nucleosides, however, carbamate linkageshave been used to form fixed length 3′ to 5′ internucleoside linkage asreported by Stirchak et al., J. Orq. Chem., 52, 4202, 1987.

While currently utilized nucleosides, nucleotides and oligonucleotidesconjugate linking moieties certainly have great utility, there is acontinuing need for improved conjugate linking groups have a potpourriof different properties. One such property is as a transitory blockinggroup during oligonucleotide synthesis. A further property is to providea foundation at the 2′ and 3′ positions where “spacer” molecules ofvarious lengths, e.g., diaminoalkyl groups such as 1,2-ethylene diamineand 1,6-diaminohexane, can be attached for modifying the spacing betweenthe nucleosides, nucleotides or oligonucleotides and the conjugategroup.

OBJECTS OF THE INVENTION

It is one object of this invention to provide nucleosides,oligonucleotides and oligonucleosides that include carbamate chemicalfunctionalities.

It is a further object of the invention to provide compounds for linkingvarious conjugate groups via carbamate linkages to nucleosides,oligonucleotides and oligonucleosides.

It is another object to provide compounds that include conjugatedintercalators, nucleic acid cleaving agents, cell surface phospholipids,and/or diagnostic agents.

It is yet another object to provide improvements in research anddiagnostic methods and materials for assaying bodily states in animals,especially disease states.

It is an additional object of this invention to provide therapeutic andresearch materials having modified or improved spectral, solubility,transfer or uptake properties for the identification and analysis of DNAand RNA, for the diagnosis of normal or disease states of cells,cellular components or organisms and treatment of diseases throughvarious mechanisms including modulation of the activity of DNA or RNA.

BRIEF DESCRIPTION OF THE INVENTION

These and other objects are satisfied by the present invention, whichprovides compounds containing carbamate chemical functionalities. In oneaspect, the invention provides oligonucleosides comprising a pluralityof linked nucleosides, each of which includes a base portion and aribofuranosyl sugar portion. In certain embodiments, at least one ofsuch nucleosides bears at a 2′-O-position or a 3′-O-position asubstituent having formula:

—R_(A)—N—C(X)—O—R_(1a)

or

—C(X)—N(R_(1b))(R_(1c))

where:

R_(A) is alkyl having from 1 to about 10 carbon atoms or(CH₂—CH₂—Q)_(x);

R_(1a) is alkenyl having 2 to about 10 carbon atoms;

R_(1b) and R_(1c), independently, are H, R₂, R_(A), an amine protectinggroup or have formula R_(A)—N(R_(1d))(R_(1e)), C(X)—R₂, C(X)—R_(A)—R₂,C(X)-Q—R_(A)—R₂, or C(X)-Q—R₂;

R_(1d) and R_(1a), independently, are H, R₂, R_(A), an amine protectinggroup or have formula C(X)—R₂, C(X)—R_(A)—R₂, C(X)-Q—R_(A)—R₂, orC(X)-Q—R₂;

R₂ is a steroid molecule, a reporter molecule, a lipophilic molecule, areporter enzyme, a peptide, a protein, includes folic acid or hasformula -Q-(CH₂CH₂-Q-)_(x)-R₃;

X is O or S;

each Q is, independently, is NH, O, or S;

x is 1 to about 200;

R₃ is H, R_(A), C(O)OH, C(O)OR_(A), C(O)R₄, R_(A)—N₃, or R_(A)—NH₂;

R₄ is Cl, Br, I, SO₂R₅ or has structure:

m is 2 to 7; and

R₅ alkyl having 1 to about 10 carbon atoms.

In other embodiments, at least one of the nucleosides of the compoundsof the invention includes a pyrimidine base portion which bears at its5-position a substituent having formula:

—R_(A)—O—C(X)—N(R_(1b))(R_(1c))

where R_(A), X, R_(1b), and R_(1c) are as defined above.

The present invention also provides methods for inhibiting theexpression of particular genes in the cells of an organism, comprisingadministering to said organism a compound according to the invention.Also provided are methods for inhibiting transcription and/orreplication of particular genes or for inducing degradation ofparticular regions of double stranded DNA in cells of an organism byadministering to said organism a compound of the invention. Furtherprovided are methods for killing cells or virus by contacting said cellsor virus with a compound of the invention. The compound can be includedin a composition that further includes an inert carrier for thecompound.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides nucleosides, oligonucleotides andoligonucleosides containing carbamate chemical functionalities. Thenucleoside subunits can be “natural” or “synthetic” moieties. Eachnucleoside is formed from a naturally occurring or synthetic base and anaturally occurring or synthetic pentofuranosyl sugar group.

The term “oligonucleotide” refers to a polynucleotide formed from aplurality of linked nucleotide units. The nucleotides units each includea nucleoside unit. In the context of this invention, the term“oligonucleoside” refers to a plurality of nucleoside units that arelinked together. In a generic sense, since each nucleotide unit of anoligonucleotide includes a nucleoside therein, the term“oligonucleoside” can be considered to be inclusive of oligonucleotides(i.e., nucleosides linked together via phosphate linking groups). In afurther sense, the term “oligonucleoside” also refers to a plurality ofnucleosides that are linked together via linkages other than phosphatelinkages. The term “oligonucleoside” thus effectively includes naturallyoccurring species or synthetic species formed from naturally occurringsubunits. For brevity, the term “oligonucleoside” will be used asencompassing both phosphate linked (oligonucleotides) and non-phosphatelinked polynucleoside species.

Oligonucleosides according to the invention also can include modifiedsubunits. Representative modifications include modification of aheterocyclic base portion of a nucleoside or a sugar portion of anucleoside. Exemplary modifications are disclosed in the following U.S.Pat. Nos. 5,138,045, 5,212,295, 5,223,618, 5,359,051, 5,359,044,5,378,825, 5,457,191, 5,459,255, 5,489,677, 5,506,351, 5,519,134,5,541,307, 5,543,507, 5,130,302, 5,134,066, 5,432,272, 5,457,187,5,484,908, 5,502,177, 5,216,141, 5,434,257 and 3,687,808. The disclosureof each of these patents is incorporated herein by reference.

The term oligonucleoside thus refers to structures that include modifiedportions, be they modified sugar moieties or modified base moieties,that function similarly to natural bases and natural sugars.Representative modified bases include deaza or aza purines andpyrimidines used in place of natural purine and pyrimidine bases;pyrimidines having substituent groups at the 5- or 6-position; andpurines having altered or replacement substituent groups at the 2-, 6-,or 8-positions. Representative modified sugars include carbocyclic oracyclic sugars, sugars having substituent groups at their 2′-position,and sugars having substituents in place of one or more hydrogen atoms ofthe sugar.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the spirit of this invention. Sucholigonucleosides are best described as being structurallydistinguishable from yet functionally interchangeable with naturallyoccurring or synthetic wild type oligonucleotides. All sucholigonucleosides are comprehended by this invention so long as theyfunction effectively to mimic the structure of a desired RNA or DNAstrand.

The compounds of the present invention are those which bear asubstituent having formula:

—R_(A)—N—C(X)—O—R_(1a)

or

—C(X)—N(R_(1b))(R_(1c))

at a 2′-O- or 3′-O-nucleoside position or which bear a substituenthaving formula:

—R_(A)—O—C(X)—N(R_(1b))(R_(1c))

at a 5-pyrimidine position.

R_(A) can be alkyl having from 1 to about 10 carbon atoms or(CH₂—CH₂-Q)_(x), where Q is NH, O, or S and x is 1 to about 200,preferably 1 to about 50. Alkyl groups are substituents corresponding tobranched and unbranched hydrocarbons. Preferred alkyl groups accordingto the invention have from 1, 2, or 6 carbon atoms.

R_(1a) can be alkenyl having 2 to about 10 carbon atoms. Preferredalkenyl groups are those having 2 to about 5 carbon atoms. Oneparticularly preferred alkenyl group is the 2-propenyl (i.e.,—CH₂CH═CH₂) group.

R_(1b) R_(1c), R_(1d), and R_(1e), independently, can be H, R₂, R_(A),an amine protecting group or have formula R_(A)—N(R_(1d))(R_(1e)),C(X)—R₂, C(X)—R_(A)—R₂, C(X)—Q—R_(A)—R₂, or C(X)—Q—R₂. In preferredembodiments, R_(1b) (and/or R_(1d)) is H and R_(1c) (and/or R_(1e)) isH, R₂, or R_(A), or R_(1b) and R_(1c) (and/or R_(1d) and R_(1e)),together, are a phthalimido amine protecting group. In otherembodiments, R_(1d) is H and R_(1e) is C(X)—Q—R₂ where X is S or,preferably, O, and Q is NH, O, or S.R_(1d) is H and R_(1e) is R₂ orC(x)—Q—R₂.

R₂ is a steroid molecule, a reporter molecule, a lipophilic molecule, areporter enzyme, a peptide, a protein, or has formula—Q—(CH₂CH₂—Q—)_(x)-R₃. In preferred embodiments, R₂ includes cholesterolor folic acid (i.e., includes a substantial portion of the cholesterolor folic acid molecule). For the purposes of this invention the terms“reporter molecule” and “reporter enzyme” are inclusive of thosemolecules or enzymes that have physical or chemical properties thatallow them to be identified in gels, fluids, whole cellular systems,broken cellular systems and the like utilizing physical properties suchas spectroscopy, radioactivity, colorimetric assays, fluorescence, andspecific binding. Steroids include those chemical compounds that containa perhydro-1,2-cyclopentanophenanthrene ring system. Proteins andpeptides are utilized in their usual sense as polymers of amino acids.Normally peptides comprise such polymers that contain a smaller numberof amino acids per unit molecule than do the proteins. Lipophilicmolecules include naturally-occurring and synthetic aromatic andnon-aromatic moieties such as fatty acids, esters, alcohols and otherlipid molecules, substituted aromatic groups such as dinitrophenylgroups, cage structures such as adamantane and buckminsterfullerenes,and aromatic hydrocarbons such as benzene, perylene, phenanthrene,anthracene, naphthalene, pyrene, chrysene, and naphthacene.

Particularly useful as steroid molecules are the bile acids includingcholic acid, deoxycholic acid and dehydrocholic acid; steroids includingcortisone, digoxigenin, testosterone and cholesterol and even cationicsteroids such as cortisone having a trimethylaminomethyl hydrazide groupattached via a double bond at the 3-position of the cortisone rings.Particularly useful as reporter molecules are biotin, dinitrophenyl, andfluoresein dyes. Particularly useful as lipophilic molecules are steroidgroups, alicyclic hydrocarbons, saturated and unsaturated fatty acids(such as palimitic and oleic), waxes, terpenes and polyalicyclichydrocarbons including adamantane and buckminsterfullerenes.Particularly useful as reporter enzymes are alkaline phosphatase andhorseradish peroxidase. Particularly useful as peptides and proteins aresequence-specific peptides and proteins including phosphodiesterase,peroxidase, phosphatase and nuclease proteins. Such peptides andproteins include SV40 peptide, RNaseA, RNase H and Staphylococcalnuclease. Particularly useful as terpenoids are vitamin A, retinoicacid, retinal and dehydroretinol.

Representative PEG groups are disclosed by Ouchi, et al., Drug Designand Discovery 1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56,4329, and Delgardo et. al., Critical Reviews in Therapeutic Drug CarrierSystems 1992, 9, 249.

For use in antisense methodology, the oligonucleosides of the inventionpreferably comprise from about 10 to about 30 subunits. It is morepreferred that such oligonucleosides comprise from about 15 to about 25subunits. As will be appreciated, a subunit is a base and sugarcombination suitably bound to adjacent subunits through, for example, aphosphorous-containing (e.g., phosphodiester) linkage or some otherlinking moiety. The nucleosides need not be linked in any particularmanner, so long as they are covalently bound. Exemplary linkages arethose between the 3′- and 5′-positions or 2′- and 5′-positions ofadjacent nucleosides. Exemplary linking moieties are disclosed in thefollowing references: Beaucage, et al., Tetrahedron 1992, 48, 2223 andreferences cited therein; and U.S. Pat. Nos. 3,687,808, 4,469,863,4,476,301, 5,023,243, 5,034,506, 5,177,196, 5,214,134, 5,216,141,5,264,423, 5,264,562, 5,264,564, 5,321,131, 5,399,676, 5,405,939,5,434,257, 5,455,233, 5,476,925, 5,470,967, 5,495,009 and 5,519,126, aswell as others of the above referenced patents. The disclosure of eachof these patents is incorporated herein by reference.

It is preferred that the RNA or DNA portion which is to be modulatedusing oligonucleosides of the invention be preselected to comprise thatportion of DNA or RNA which codes for the protein whose formation oractivity is to be modulated. The targeting portion of the composition tobe employed is, thus, selected to be complementary to the preselectedportion of DNA or RNA, that is, to be an antisense oligonucleoside forthat portion.

In accordance with one preferred embodiment of this invention, thecompounds of the invention can be targeted to various mRNA sequencesincluding those disclosed in U.S. Pat. Nos. 5,166,195, 5,242,906,5,248,670, 5,442,049, 5,457,189, 5,510,239, 5,514,577, 5,514,788,5,539,389 and 5,530,114. The disclosure of each of these patents isincorporated herein by reference.

The nucleosides and oligonucleosides of the invention can be used indiagnostics, therapeutics and as research reagents and kits. They can beused in pharmaceutical compositions by including a suitablepharmaceutically acceptable diluent or carrier. They further can be usedfor treating organisms having a disease characterized by the undesiredproduction of a protein. The organism should be contacted with anoligonucleotide having a sequence that is capable of specificallyhybridizing with a strand of nucleic acid coding for the undesirableprotein. Treatments of this type can be practiced on a variety oforganisms ranging from unicellular prokaryotic and eukaryotic organismsto multicellular eukaryotic organisms. Any organism that utilizesDNA-RNA transcription or RNA-protein translation as a fundamental partof its hereditary, metabolic or cellular control is susceptible totherapeutic and/or prophylactic treatment in accordance with theinvention. Seemingly diverse organisms such as bacteria, yeast,protozoa, algae, all plants and all higher animal forms, includingwarm-blooded animals, can be treated. Further, since each cell ofmulticellular eukaryotes can be treated since they include both DNA-RNAtranscription and RNA-protein translation as integral parts of theircellular activity. Many of the organelles (e.g., mitochondria andchloroplasts) of eukaryotic cells also include transcription andtranslation mechanisms. Thus, single cells, cellular populations ororgarelles can also be included within the definition of organisms thatcan be treated with therapeutic or diagnostic oligonucleotides. As usedherein, therapeutics is meant to include the eradication of a diseasestate, by killing an organism or by control of erratic or harmfulcellular growth or expression.

In preparing compounds of the invention, one or more protecting groupscan be used for temporary blocking a chemically reactive site in themolecules. Protecting groups are known per se as chemical functionalgroups that can be selectively appended to and removed fromfunctionalities, such as amine groups. These groups are present in achemical compound to render such functionality inert to chemicalreaction conditions to which the compound is exposed. See, e.g., Greeneand Wuts, Protective Groups in Organic Synthesis, 2d edition, John Wiley& Sons, New York, 1991. Numerous amine protecting groups are known inthe art, including, but not limited to: phthalimide (PHTH),trifluoroacetate (triflate), allyloxycarbonyl (Alloc), benzyloxycarbonyl(CBz), chlorobenzyloxycarbonyl, t-butyloxycarbonyl (Boc),fluorenylmethoxycarbonyl (Fmoc), and isonicotinyloxycarbonyl (i-Noc)groups. (see, e.g., Veber and Hirschmann, et al., J. Org. Chem. 1977,42, 3286 and Atherton, et al., The Peptides, Gross and Meienhofer, Eds,Academic Press; New York, 1983; Vol. 9 pp. 1-38).

Oligonucleosides according to the invention can be assembled in solutionor through solid-phase reactions, for example, on a suitable DNAsynthesizer utilizing nucleosides according to the invention and/orstandard nucleotide precursors. The nucleosides and nucleotideprecursors can already bear alkylamino groups or can be later modifiedto bear such groups. Suitably protected nucleosides can be assembledinto an oligonucleosides according to known techniques. See, e.g.,Beaucage, et al., Tetrahedron 1992, 48, 2223.

Oligonucleosides according to the invention also can be prepared byassembling an oligonucleoside and appending an appropriate functionalitythereto. For example, oligonucleosides having free hydroxyl groups canbe assembled according to known techniques and then reacted with areagent for linking the appropriate carbamate group thereto. As will berecognized, however, greater selectivity can be achieved in terms ofplacement of carbamate functionality within an oligonucleoside byintroducing such functionality, as discussed above, on selectednucleosides and then using both the selected nucleosides and othernucleosides to construct an oligonucleoside.

Thus, the invention first builds the desired linked nucleoside sequencein the normal manner on the DNA synthesizer. One or more (preferably twoor more) of the linked nucleosides are then functionalized orderivatized with the lipophilic steroid, reporter molecule, lipophilicmolecule, reporter enzyme, peptide or protein.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples, which are not intended to be limiting. Alloligonucleotide sequences are listed in a standard 5′ to 3′ order fromleft to right.

EXAMPLE 12′-O-(N-alloc-6-aminohexyl)-5′-O-dimethoxytrityl-5-methyluridine(Compound 1)

2′-O-(6-Aminohexyl)-5′-O-dimethoxytrityl-5-methyluridine (1.98 g, 3mmole, synthesized genarally according to the procedure of Manoharan, etal., Tetrahedron Lett. 1995, 36, 3647 and Tetrahedron Lett. 1995, 36,3651 using 5-methyl uridine in place of uridine) was coevaporated twicewith anhydrous pyridine (2×25 mL) and the residue was dissolved in 30 mLof dry pyridine. Allyl-1-benzotriazolyl carbonate (720 mg, 3.3 mmole)was added to the pyridine solution and the reaction mixture was stirredat room temperature. Thin layer chromatography (TLC) analysis(CH₃OH:CH₂Cl₂ 1:9) after 15 min. indicated the reaction was complete.The solvent was evaporated and the residue was purified by silica gelcolumn chromatography using 2% CH₃OH in CH₂Cl₂. The yield was about 0.99g (90%) . The N-alloc protected compound was obtained as a white foam.R_(f)=0.6 in CH₃OH:CH₂Cl₂ 1:9.

EXAMPLE 2 Deprotection of Alloc Group at the Nucleoside Stage

The N-alloc compound prepared in Example 1 (75 mg, 0.1 mmol) wasdissolved in 10 mL dry tetrahydrofuran (THF). To this solution 87 μL ofmorpholine (1 mmol) was added followed bytetrakis(triphenylphosphine)palladium (0) 15 mg (0.013 mmol). Thereaction flask was covered with aluminum foil to protect it from lightand was stirred. The reaction was followed by TLC analysis. After 6hours, the reaction was complete, generating the amino nucleoside.

EXAMPLE 32′-O-(N-Allyloxycarbonyl-6-aminohexyl)-5′-O-dimethoxytrityl-5-methyluridine-3′-O-(2-cyanoethyl)-N,N-diisopropylPhosphoramidite (Compound 2 (T₂*Alloc Phosphoramidite))

2-O-(N-allyloxycarbonyl-6-aminohexyl)-5′-O-dimethoxytrityl-5-methyluridine(3 g, 4 mmol) was dissolved in 50 mL of anhydrous CH₂Cl₂. To thissolution, 345 mg of diisopropylammonium tetrazolide (2 mmol) was addedfollowed by 1.77 mL (5.0 mmol) of 2-cyanoethyl-N,N,N′, -tetraisopropylphosphorodiamidite. The reaction mixture was stirred at room temperatureunder argon atmosphere overnight. After 16 hours, TLC(hexane:ethylacetate 4:6) indicated conversion of the nucleoside intophosphoramidite. The reaction mixture was evaporated and the residue wasapplied onto a silica gel column and eluted with hexane:ethylacetate 4:6(R_(f)=0.42). The product (2.3 g, 63%) showed two peaks around 154 ppmas expected for the phosphoramidite.

EXAMPLE 43′-O-(N-Allyloxycarbonyl-6-aminohexyl)-5′-O-dimethoxytrityl-uridine(Compound 3)

3′-O-(6-Aminohexyl)-5′-O-dimethoxytrityl-uridine (6.46 g, 10 mmol,produced generally according to the procedure of Manoharan, et al.,Tetrahedron Lett. 1995, 36, 3651) was coevaporated with dry pyridine(2×100 mL). The residue was dissolved in 100 mL of dry pyridine and tothis solution allyl-1-benzotriazolyl carbonate (2.4 g, 11 mmol) wasadded and the reaction mixture was stirred for 1 hour. Pyridine was thenevaporated and the residue was purified on a silica gel column using 2%CH₃OH in CH₂Cl₂. The fractions containing the derived product werecombined and evaporated to give a colorless foam (3.285 g, 45% yield).R_(f)=0.53 in CH₃OH:CH₂Cl₂ 9:1.

EXAMPLE 5

3′-O-(N-Allyloxycarbonyl-6-aminohexyl)-5′-dimethoxytrityl-uridine-2′-O-succinyl-controlledPore Glass (Compound 4 (U₃*Alloc CPG))

In a 50 mL pear shaped flask 2.5 g succinylated control pore glass(CPG), 0.5 g of 3′-O-(N-allyloxycarbonyl-6-aminohexyl)-5′-dimethoxyuridine (0.69 mmol), 300 mg of dimethylaminopyridine, 800 mg of1-(3-dimethylaminopropyl) ethyl carbodiimide (EDC), 20 mL of drypyridine, and 1 mL of dry triethylamine were added and the flask wasshaken in a wrist-action shaker for 24 hours. The solution was thenfiltered off and the CPG was washed with CH₂Cl₂, CH₃OH, CH₂Cl₂ and thenwith ether. It then was dried and transferred into a flask followed bythe addition of 1.3 g pentachlorophenol, 300 mg of DMAP, 0.9 g of EDC,20 mL of pyridine, and 0.5 mL of triethylamine. The flask was thenshaken for 16 hours followed by the addition of 2.5 mL of piperidine.Shaking was continued for another 2 minutes during which period anintense yellow color developed in the reaction mixture. The CPG the wasrecovered by filtration, washed with pyridine, CH₂Cl₂, CH₃OH, CH₂Cl₂and, finally, with ether. The CPG was then dried on a dessicator overP₂O₅. A 3.5 mg portion of the CPG was transferred to a 10 mL volumetricflask. The loading on the CPG was determined to be 40 μmol/gm byaddition of 3% trichloroacetic acid in CH₂Cl₂ and generating a tritylorange color.

EXAMPLE 6 Oligonucleotide Synthesis with T₂*Alloc Phosphoramidite andU₃*Alloc CPG

The following oligonucleotides were synthesized using the T₂*allocphosphoramidite and U₃*alloc CPG.

Oligomer I: TTT TTT TTT U₃* (SEQ ID NO:1) Oligomer II: TTT TT₂*T TTT T(SEQ ID NO:2) Oligomer III: T₂*GC ATC CCC CAG GCC ACC CU₃* (SEQ ID NO:3)Oligomer IV: T₂*CA GU₃* Oligomer V: T₂*GC ATC CCC CAG GCC ACC AT (SEQ IDNO:4)

Oligonucleotide phosphorothioates were synthesized on an Expeditesynthesizer on a 4×1 μmol scale. The coupling time was extended for anadditional minute. A double coupling step was employed when the modifiedamidite (Compound 2) or CPG (Compound 4) was used in the synthesiscycle. Trityl monitoring of the synthesis showed excellent couplingyields. The oligomers were not deprotected from CPG at this point.

EXAMPLE 7 Deprotection of a Allyloxycarbonyl Group in the Solid Support

The CPG column containing 1 μmol of the synthesized oligomer I wastransferred into a 5 mL pyrex screw-capped test tube. To this supportwere added 25 mg of Pd₂[Ph CH=CH₂)₂.CHCl₃, Ph₃P (64 mg) and 1 mL ofn-butyl ammoniumformate (1.2M in THF prepared generally according to theprocedure of Hayakawa, et al., Nucleosides & Nucleotides 1994, 13,1337). The test tube was capped and heated for 1.5 hour at 50° C. duringwhich the solution turned black. The supernatant then was discarded andthe CPG was washed extensively with THF, acetone, a solution of sodiumN,N-diethyl-dithio carbamate (SDDTC, 0.1M in water, pH 9.78) for 10min., acetone, water, acetone, SDDTC, water, acetone, CH₂Cl₂ and ether.

An aliquot of the alloc-deprotected CPG (10 mg) was placed in anotherpyrex screw-capped vial and deprotected with concentrated ammonia at 55°C. for 2 hours. The ammonia solution was evaporated to dryness in aspeed vac and the residue was dissolved in 1 mL of water, filtered bycentrifugation using a nylon-66 0.45 μ filter.

The resulting oligonucleotide was analyzed by analytical HPLC and massspectrometry.

The other oligonucleotides (Oligomers II-V) were also deprotectedaccording to the same protocol.

EXAMPLE 8 Conjugation of Pyrene Butyric Acid to Oligomer I on the SolidSupport

Oligomer I-CPG (from which the allyloxy the allyloxycarbonyl group wasremoved in the solid support) was suspended in 2.5 mL ofdimethylsulfoxide/pyridine (8:2, v/v). Pyrene butyricacid-N-hydroxysuccinimide ester (and 50 mg) was added to it in a reactorand the resulting mixture was shaken overnight. The DMSO/pyridinesolution was filtered off and the CPG was washed with DMSO, CH₂Cl₂, andether and then dried. The CPG then was deprotected in concentratedammonia (2 mL) at 55° C. for 2 hours. The ammonia solution was thencooled and evaporated. The residue was dissolved in 1 mL of water andfiltered. UV-vis spectral analysis indicated formation of pyreneconjugate which was confirmed by HPLC and mass spectral analysis.

EXAMPLE 9 HPLC and Mass Spectral Analysis of Oliogmer I Derivatives

Retention Mass Spec. Modification Time¹ Expected Observed Oligomer I34.7 3225.51  3225.01 3′-O-(6-aminohexyl) Oligomer I 39.1 3309.55 3308.43′-O-(N-alloc-6-aminohexyl) Oligomer I 52.3 3495.86 3497.03′-O-(N-pyrene butyrate-6-aminohexyl) ¹HPLC conditions: Waters 600E with991 detector; Waters Delta Pak C-18 column (3.9 × 300 mm) Solvent A: 50mm TEAAc pH 7.0; B: 100% CH₃CN. 1.5 mL/min flow rate. Gradient: 5% B forfirst 10 minutes with linear increase in B to 40% during the next 50minutes.

EXAMPLE 10 Solution Phase Conjugation of Pyrenebutyric Acid to OligomerII

Oligonucleotide II-CPG was deprotected as in Example 7 for alloc groupin CPG and then in NH₄OH to cleave the oligonucleotide from the solidsupport. The resultant oligonucleotide was dried and dissolved in 0.2MNaHCO₃ buffer (300 μL) and to this 50 mg of pyrene butyricacid-N-hydroxysuccinimide in 350 μl of DMF was added and the solutionwas allowed to stand at room temperature overnight. The solution wasthen passed through a Sephadex G-25 column to remove the excess pyrenereagent. The oligonucleotide was then analyzed by HPLC and mass spectralmethods to establish the formation of pyrene conjugate.

HPLC and Mass Spectral Analysis of Oligmer II and its Conjugates

HPLC Retention Mass Spec. Modification Time¹ Expected Observed OligomerII 34.64 3239.54 3240.21 2′-amine Oligomer II 38.05 3323.58 3322.042′-alloc amine Oligomer II 47.10 3509.89 3507.18 2′-pyrene ¹HPLCconditions: Waters 600E with 991 detector; Waters Delta Pak C-18 column(3.9 × 300 mm) Solvent A: 50 mm TEAAc pH 7.0; B: 100% CH₃CN. 1.5 mL/minflow rate. Gradient: 5% B for first 10 minutes with linear increase in Bto 40% during the next 50 minutes.

EXAMPLE 11 Conjugation of Cholesterol to Oligomer V in the Solid Support

Oligomer V-CPG (1 μmol) column is deprotected in the solid support asdescribed in Example 7 and the solid support is washed once withpyridine/DMSO 2:8. Then 2.5 mL of DMSO/pyridine (8:2) is added to theCPG beads followed by 100 mg of cholesterol chloroformate. The solidsupport is shaken for 2 hours. Filtering the solvents, washing withCH₂Cl₂, CH₃OH, CH₂Cl₂ and ether gives the cholesterol modifiedoligonucleotide still bound to the CPG. It is then dried and deprotectedwith concentrated NH₄OH to give the cholesterol conjugate.

EXAMPLE 123′,5′-O-Tetraisopropyl-disiloxane-1,3-diyl-N⁴-benzoylcytidine-2′-O-(Carbonyloxysuccinimide)(Compound5)

3′,5′-O-Tetraisopropyl disiloxane-1,3-diyl-N⁴-benzoylcytidine wassynthesized from N⁴-benzoyl cytidine by treatment with1,3-dichloro-1,1,3,3-tetraisopropyl 1,3-disiloxane and pyridine.N⁴-benzoylcytidine (Chem-Impex, Wood Dale, Ill.) (5 g, 14.4 mmol) wascoevaporated with pyridine (2×50 mL) and treated with 50 mL anhydrouspyridine and 5 g of 1,3-dichloro-1,1,3,-tetraisopropyl-1,3,disiloxane(15.85 mmol) under argon atmosphere. After 4 hours pyridine wasevaporated and the residue was partitioned between CH₂Cl₂ and saturatedNaHCO₃ solution. The organic layer was washed once with saturated NaClsolution and evaporated to give crude product of 3′-5′-O-tetraisopropyldisiloxane-1,3-diyl-N⁴ benzoyl cytidine. The material was purified in asilica column (9:1 CH₂Cl₂/CH₃OH) and eluted to give the pure compound(7.9 g, 93%) as a white foam.

3′,5′-O-TIPS-N⁴-benzoyl cytidine (5 g, 8.48 mmols) was dissolved in 25mL of anhydrous acetonitrile and 25 mL methylene chloride. To thissuspension N,N′-disuccinimidyl carbonate (3.6 g, 14 mmols) andtriethylamine (25 mmol, 3.5 mL) were added. The resulting suspension wasshaken in a wrist-action shaker until no starting material remained byTLC. The mixture was concentrated under reduced pressure and the residuewas diluted with aqueous saturated NaHCO₃ solution (200 mL) andextracted thoroughly with methylene chloride (2×100 mL). The combinedextracts were washed with saturated NaCl solution and dried overmagnesium sulfate. Evaporation of the methylene chloride solutionyielded the mixed carbonate which was used in further chemistry withoutany further purification (6.5 g yield).

EXAMPLE 13 Cholesteryl-oxycarbonyl-aminohexylamine (Compound 6)

Cholesteryl chloroformate (Fluka, 11.3 g, 25 mmol) was dissolved in 100mL of anhydrous methylene chloride. This was added dropwise to 20 g (172mmol) of 1,6-hexanediamine taken in 250 mL of pyridine:methylenechloride (1:1 v/v). The reaction mixture was stirred for 2 hours afterwhich it was evaporated and extracted between methylene chloride (100mL) and saturated NaHCO₃ solution (100 mL). The organic layer was washedone more time with saturated NaHCO₃ solution followed by saturated NaClsolution, dried over anhydrous K₂CO₃, and evaporated to give the desiredcompound as a yellow waxy compound. ¹³C NMR showed (CDCl₃) onehomogeneous compound, exhibiting both cholesterol and hexylamine carbonresonances.

EXAMPLE 142,′-O-(Carbonylamino-hexylamino-carbonyl-oxycholesteryl-3′-5′-O-TIPS-N⁴-benzoylCytidine (Compound 7)

3,5′-TIPS-N⁴-benzoyl-2′-O-(carbonyloxysuccinimidyl) cytidine (1 g, 1.41mmol) was dissolved in CH₂Cl₂ (3 mL). To this solution was addedcholesteryl-oxycarbonylaminohexylamine (1 g, 1.9 mmol) in methylenechloride (5 mL) containing triethylamine (2.2 mmol, 0.3 mL) and pyridine(0.5 mL) with stirring. The resulting mixture was stirred at roomtemperature until no mixed carbonate remained by TLC (4 hours). Thereaction mixture was then diluted with CH₂Cl₂ (50 mL) and washedsuccessively with saturated aqueous NaHCO₃ solution (50 mL), saturatedNaCl solution and dried over MgSO₄. Removal of the solvent, followed bychromotography over silica gel 4:6 ethylacetate/hexanes afforded thecarbamate (1.14 g, 0.99 mmol) in 70% yield.

EXAMPLE 15 5′-O-Dimethoxytrityl-2′-O-(carbonylaminohexylaminocarbonyloxy cholesteryl)-N⁴-benzoyl Cytidine (Compound 8

3′-5′O-TIPS-2′-O-(carbonylamino-hexylamino carbonyloxycholesteryl)-N⁴-benzoyl-cytidine is treated with (nBu)₄NF in pyridine.The resultant 3′-5′-dihydroxy compound is treated with dimethoxytritylchloride/pyridine to give the corresponding 5′-O-dimethoxytritylcompound.

EXAMPLE 165′-O-(Dimethoxytrityl)-2′-O-[(carbonyl-aminohexyl-aminocarbonyl-oxy-cholesteryl]N⁴-benzoyl-cytidine-3′-O-[2-cyanoethyl-N,N-diisopropyl]phosphoramidite(Compound 9)

5′-O-(Dimethoxytrityl)-2′-O-[carbonylaminohexyl-aminocarbonyloxy-N-(3-oxycarbonyl-cholesteryl)amino]N⁴-benzoylcytidine is dissolved in dry dichloromethane. 2-CyanoethylN,N,N′N′-tetraisopropylphosphorodiamidite and diisopropylammoniumtetrazolide are added to the mixture, which is stirred under argon for16 hours. Dichloromethane is added to the solution, washed with an equalvolume of saturated NaHCO₃. The aqueous layer is washed with an equalvolume of dichloromethane. The combined organic layers are washed withan equal volume of saturated NaCl and dried over MgSO₄ and concentratedin vacuo. The residue is chromatographed on a silica gel column with agradient of 25% ethyl acetate in hexanes to 70% ethyl acetate to yieldthe amidate.

EXAMPLE 175′-O-(Dimethoxytrityl)-2′-O-[carbonylaminohexyl-N-(3-oxycarbonyl-cholesteryl)amino]N⁴-O-benzoyl-cytidine-3′-O-(succinylaminopropyl)-Controlled Pore Glass (Compound 10)

Succinylated and capped controlled pore glass (0.3 grams) is added to2.5 ml anhydrous pyridine in a 15 ml pear-shaped flask. DEC (0.07 grams,0.36 mmol), TEA (100 μl, distilled over CaH₂), DMAP (0.002 grams, 0.016mmol) and5′-O-(dimethoxytrityl)-2′-O-[carbonylaminohexyl-N′-(3-oxycarbonyl-cholesteryl)amino]N⁴-benzoylcytidine are added under argon and the mixture shaken mechanically for16 hours. More nucleoside (0.20 grams) is added and the mixture shakenan additional 18 hours. Pentachlorophenol (0.03 grams, 0.11 mmol) isadded and the mixture shaken 9 hours. CPG is filtered off and washedsuccessively with dichloromethane, triethylamine, and dichloromethane.CPG is then dried under vacuum, suspended in 10 ml piperidine and shaken5 minutes. CPG is filtered off, washed thoroughly with dichloromethaneand again dried under vacuum. The extent of loading is determined byspectrophotometric assay of dimethoxytrityl cation in 0.2 Mp-toluenesulfonic acid at 498 nm as approximately 31 μmol/g.

EXAMPLE 18 Oligonucleotide Synthesis

Oligonucleotides incorporating cholesterol carbamate nucleoside aresynthesized in an Expedite Synthesizer. An extended coupling time isused for cholesterol building blocks.

Oligomer TGC* ATC CCC CAG GCC ACC AT VI (P = S) Oligomer TC*G CAT CGACCC GCC CAC TA (SEQ ID NO:5) VII (P = S) C* = Compound 9 synthesizedaccording to Example 16

The oliognucleotides are purified by standard HPLC protocols.

EXAMPLE 19 Assay for ICAM-1 Using 2′-Cholesterol ConjugatedOligonucleotides Materials

Cholesterol-oligonucleotide assays for ICAM-1 are done in the bEnd.3cell line, a brain endothelioma using Opti-MEM, trypsin-EDTA and DMEMwith high glucose (all from Gibco-BRL, Grand Island, N.Y.), Dulbecco'sPBS (Irvine Scientific, Irvine, Calif.), sterile, 12 well tissue cultureplates and Facsflow solution (from Becton Dickinson, Mansfield, Mass.),ultrapure formaldehyde (Polysciences, Warrington, Pa.), recombinanthuman TNF-α (R&D Systems, Minneapolis, Minn., mouse interferon-γ(Genzyme, Cambridge, Mass.), and Fraction V, BSA (Sigma, St. Louis,Mo.). The mouse ICAM-1-PE, VCAM-1-FITC, hamster IgG-FITC and ratIgG2α-PE antibodies were purchased from Pharmingen (San Diego, Calif.).Zeta-Probe nylon blotting membrane was purchased from Bio-Rad (Richmond,Calif.). QuickHyb solution was purchased from Stratagene (La Jolla,Calif.). A cDNA labeling kit, Prime-a-Gene, was purchased from ProMega(Madison, Wis.). NAP-5 columns were purchased from Pharmacia (Uppsala,Sweden).

Oligonucleotide Treatment

Cells are grown to approximately 75% confluency in 12 well plates withDMEM containing 4.5 g/L glucose and 10% FBS. Cells are washed 3 timeswith Opti-MEM pre-warmed to 37° C. Oligonucleotide VI is premixed withOpti-MEM, serially diluted to desired concentrations and transferredonto washed cells for a 4 hour incubation at 37° C. Media is removed andreplaced with normal growth media with or without 5 ng/ml TNF-α and 200U/ml interferon-γ, incubated for 2 hours for northern blot analysis ofmRNA or overnight for flow cytometric analysis of cell surface proteinexpression.

Flow Cytometry

After oligonucleotide treatment, cells are detached from the plates witha short treatment of trypsin-EDTA (1-2 min.). Cells are transferred to12×75 mm polystyrene tubes and washed with 2% BSA, 0.2% sodium azide inD-PBS at 4° C. Cells are centrifuged at 1000 rpm in a Beckman GPRcentrifuge and the supernatant is then decanted. ICAM-1, VCAM-1 and thecontrol antibodies are added at 1 ug/ml in 0.3 ml of the above buffer.Antibodies are incubated with the cells for 30 minutes at 4° C. in thedark, with gentle agitation. Cells are washed again as above and thenresuspended in 0.3 ml of FacsFlow buffer with 0.5% ultrapureformaldehyde. Cells are analyzed on a Becton Dickinson FACScan. Resultsare expressed as percentage of control expression, which is calculatedas follows: [((CAM expression for oligonucleotide-treated cytokineinduced cells)−(basal CAM expression))/((cytokine-induced CAMexpression)−(basal CAM expression))]×100. For the experiments involvingcationic lipids, both basal and cytokine-treated control cells arepretreated with Lipofectin for 4 hours in the absence ofoligonucleotides. (Bennett, et al., Mol Pharmacol. 1992, 41, 1023.)

RNA Isolation and Analysis

Total cellular RNA is isolated by cellular lysis in 4M guanidiniumisothiocyanate followed by a CsCl gradient. Total cellular RNA isseparated on a 1.2% agarose gel containing 1.1% formaldehyde, thentransferred to the nylon membrane and UV crosslinked to the membraneusing a Stratagene UV crosslinker 2400. Blots are hybridized with cDNAprobes purified on NAP-5 columns that are random primed for 1 to 2 hoursin QuickHyb solution. Blots are washed 2 times at 25° C. in 2×SSC with0.1% SDS for 10 minutes each and then washed 1 time in 0.1% SSC with0.1% SDS at 60° C. for 30 minutes.

EXAMPLE 20 Effect of 2′-Cholesterol Conjugated Oligonucleotide on ICAM-1Expression

The nucleoside-cholesterol conjugate (Compound 9) from Example 16 isincorporated into the antisense oligonucleotide developed for mousemodel studies (ISIS 3082) disclosed by Stepkowski, et al., J. Immunol.1994, 5337.)

Oligomer VI TGC* ATC CCC CAG GCC ACC AT (P = S)

The resultant conjugate (Oligomer VI) is tested for inhibiting ICAM-1expression. ISIS-3082 shows antisense inhibition in cell culture with anIC₅₀ of 100 nM when formulated with a cationic lipid for delivery.

In cell culture comparison experiments evaluating the effect ofISIS-3082 and Oligomer VI on controlling ICAM-1 expression without anycationic lipid adjuvant, oligomer VI inhibits ICAM-1 in a dose dependentmanner. ISIS-3082 does not show any activity at all, even when highconcentrations are used. Furthermore, the inhibition of proteinexpression appears to be target specific. When analyzed for controllingthe isotype protein VCAM-1, neither molecule show significant inhibitionof VCAM-1 expression. Since no sequence similarity exists between themouse ICAM-1 sequence and the mouse VCAM-1 sequence, ISIS-3082 or itsconjugate would not be expected to influence the expression of VCAM-1 ifthey are working through an antisense mechanism.

EXAMPLE 21 Cholesterol Conjugation Affects the Biodistribution of theOligonucleotide

The effect of cholesterol conjugation on the pharmacokinetic propertiesof the oligonucleotide is determined in mice using ³H radiolabeledOligonucleotide VI. This modification shows a marked influence on thebiodistribution of the oligonucleotide. ISIS-3082 is mainly distributedin liver, kidney, skeletal muscle and skin. In the case of Oligomer VI,more oligonucleotide is found in the liver. The amount is reduced inkidney, skeletal muscle and skin. Oligomer VI is also retained in theplasma for longer periods of time than is ISIS-3082 which is consistentwith the improved efficacy of Oligomer VI.

EXAMPLE 22 Inhibition of ICAM-1 Expression in Mouse Liver UsingCholesterol Conjugated Oligonucleotides

Eight to twelve week old C57Bl/10 mice are injected twice intravenouslywith 10 mg/kg oligonucleotide at 24 hours and 2 hours beforelipopolysaccharide (LPS) administration. A 25 ugs portion of LPS from S.typhosa (Difco Labs, Detroit, Mich.) is injected intraperitoneally andthe mice are sacrificed 4 hours later. Total RNA from the liver isisolated and analyzed as described above. To determine if the increaseddelivery of oligonucleotides to liver correlated with increasedefficacy, mice are treated with the ICAM-1 antisense oligonucleotidesand then ICAM-1 expression is induced by treating with bacterialendotoxin (Lipopolysaccharides, LPS). Oligomer VI at a dose of 10 mg/kgreduces ICAM expression by 40-50% while unconjugated oligonucleotidefailed to affect ICAM-1 expression. More importantly, increasing thedose of ISIS-3082 up to 100 mg/kg did not result in significantinhibition.

EXAMPLE 23 Correlation of Stability with Activity and Sequence SpecificActivity of Oligonucleotides

The following compounds are evaluated: (I) deoxyphosphorothioateoligomer ISIS-3082; (ii) Oliogmer VI; (iii) Oligomer VII; and (iv)Oligomer VIII.

Two sets of in vitro experiments are carried out with these compounds:assay of ICAM-1 mRNA by Northern blot analysis and ICAM-1 proteincell-surface expression by FACS analysis in bEnd.3 cells. In proteininhibition, ISIS-3082 complexed with cationic lipids inhibited ICAM-1expression with an IC₅₀ at 100 nm concentration. In the absence ofcationic lipids, neither ISIS-3082 by itself, Oligomer VII, nor OligomerVIII, cholesterol conjugate thereof, reduces ICAM-1 gene expression.These results establish that effects are sequence specific and prove theantisense mechanism of action.

EXAMPLE 24 3′,5′,-O-TIPS-N⁴-Benzoyl-2′-O-(Carbonylamino EthyleneDiamino)Cytidine (Compound 11)

3′,5′-TIPS-N⁴-benzoyl-2′-O-(carbonyloxy succinimidyl) cytidine (1 g,1.41 mmol) was dissolved in CH₂Cl₂ (3 mL). To this solution 1.5equivalents of ethylene diamine in CH₂Cl₂ was added very slowly withexternal cooling. A white turbidity was noticed. After 4 hours thesolution was evaporated, separated between saturated NaHCO₃ solution andCH₂Cl₂ layers. The organic layer was washed with saturated NaCl solutiondried over anhydrous magnesium sulfate and evaporated. The ¹³C NMR ofthe residue showed an ethylene diamine unit connected to 2′-position viaa carbamate linkage.

EXAMPLE 25 N-(Phthalimido)ethylenediamine (PHTH-NH—CH₂—CH₂—NH₂)(Compound 12)

One equivalent of phthalic anhydride is treated with 10 equivalents ofethylenediamine in CH₂Cl₂ under high dilution conditions. After checkingfor the product in TLC, the reaction mixture is evaporated and workedup. The excess ethylene diamine is removed with aqueous layer leavingthe monoalkylated product.

EXAMPLE 265′-O-Dimethoxytrityl-N⁴-benzoyl-2′-O-[carbonylamino-N-phthalimidoethylenediamine]-3′-O-(2-cyanoethyl-N,N-diisopropyl) Phosphoramidite(Compound 13)

3′,5′-TIPS-N⁴-benzoyl-2′-O-(carbonyloxy succinimidyl) cytosine istreated with N-phthalimido ethylenediamine. The resultant carbamate istreated with TBAF in THF to give the 3′,5′-dihydroxycompound which isthem dimethoxytritylated at the 5′-position withdimethoxytritylchloride/pyridine. The 5′-protected nucleoside is thenphosphitylated as in Example 3.

EXAMPLE 275′-O-Dimethoxytrityl-N⁴-benzoyl-2′-O-[carbonylamino-N-phthalimidoethylenediamine]-5′-O-[succinyl-aminopropyl] Controlled Pore Glass(Compound 14)

The 5′-O-dimethoxytrityl-nucleoside of Example 26 is attached tocontrolled pore glass as in Example 5 (rather than phosphitylated) togive the desired CPG.

EXAMPLE 285′-Dimethoxytrityl-N⁶-benzoyl-2′-O-(carbonylaminoethylene(N-phthalimidodiamine) Adenosine-3′-O-[2-cyanoethyl-N,N′-diisopropyl] Phosphoramidite(Compound 15)

5′-dimethoxytrityl-N⁶-benzoyl-3′-t-butyldimethylsilyl adenosine(Chem-Impex International, Wood Dale, Ill.) is converted to itssuccinimidyl carbonate at the 2′-position as in Example 14. Then the3-t-butyl-dimethylsilyl group is deprotected with (n-Bu)₄NF in pyridine.The resultant nucleoside is phosphitylated as in Example 16 to give thetitle compound.

EXAMPLE 295′-O-Dimethoxytrityl-N⁶-benzoyl-2′-O-(carbonylaminoethylene(N-phthalimidodiamine) Adenosine-3′-O-(succinylaminopropyl)-Controlled Pore Glass(Compound 16)

5′-O-dimethoxytrityl-N⁶-benzoyl-3′-t-butyldimethylsilyl adenosine(Chem-Impex International, Wood Dale, Ill.) is converted to itssuccinimidyl carbonate at the 2′-position as in Example 14. Then the3′-t-butyl-dimethylsilyl group is deprotected with (NBu)₄NF in pyridine.The resultant nucleoside is converted to the corresponding controlledpore glass as in Example 17 to give the title compound.

EXAMPLE 305′-O-Dimethoxytrityl-N⁶-benzoyl-3′-O-[carbonylaminoethylene(N-phthalimido)diamine]Adenosine-2′-O-(succinylaminopropyl)-Controlled Pore Glass (Compound 17)

5′-O-dimethoxytrityl-N⁶-benzoyl-2′-t-butyldimethylsilyl adenosine(Chem-Impex International, Wood Dale, Ill.) is converted to itssuccinimidyl carbonate at the 3′-position as in Example 14. Then the2′-t-butyldimethylsilyl group is deprotected with (NBu)₄NF in pyridine.The resultant nucleoside is attached to succinylated controlled poreglass as in Example 17.

EXAMPLE 315′-O-Dimethoxytrityl-5-O-[methylene-oxy-carbonylaminoethyl(N-phthalimido)Amine]-2′-deoxyuridine (Compound 18)

5′-O-dimethoxy-5′-O-(hydroxy methyl) 2′-deoxyuridine (available fromChemGenes, Waltham, Mass.) is converted to 3′-benzoyl-5-O-(succinimidylcarbonate) by treatment with N,N-disuccinimidyl carbonate (as describedin Example 12) and by further protection of the 3′-position. Theresulting mixed carbonate is treated with 1.5 equivalents ofN-phthalimido-amino ethyl amine (Phth-NH—CH₂—CH₂—NH₂) in CH₂Cl₂containing triethylamine and pyridine. The reaction mixture is worked upas described in Example 14 to give the title compound.

EXAMPLE 325′-O-Dimethoxytrityl-5-O-[methylene-oxy-carbonylaminoethyl(N-phthalimido)Amino]-2′-deoxyuridine-3′-O-[(cyanoethyl)-N,N-diisopropyl]Phosphoramidite (Compound 19)

The nucleoside of Example 31 is phosphitylated using2-cyanoethyl-N,N,N′,N ′-tetraisopropyl phosphorodiamidite anddiisopropylammonium tetrazolide in CH₂Cl₂ solvent as described inExample 16. The phosphoramidite is purified in a silica column using50:50 ethylacetate hexanes.

EXAMPLE 335′-O-Dimethoxytrityl-5-O-[methylene-oxy-carbonylaminoethyl(N-phthalimido)Amino]-2′-deoxyuridine-3′-O-(succinylaminopropyl)-ControlledPore Glass (Compound 20)

The nucleoside from Example 30 is attached to succinylated CPG asdescribed in Example 17. The loading is determined to be 36 μMols/g.

EXAMPLE 34 2′-Carbamate-amine Linking Group Containing PhosphodiesterOligonucleotides

Compound 15 was utilized in the DNA synthesizer as a 0.1M solution inanhydrous CH₃CN. Oligonucleotide synthesis was carried out in either anABI 394 synthesizer employing the standard synthesis cycle with anextended coupling time of 10 minutes during coupling of Compound 15 intothe oligonucleotide dequence. Coupling efficiency of >98% was observedfor the coupling of the modified amidite.

The following oligonucleotides having phosphodiester inter-nucleotidelinkages were synthesized:

Oligomer CTG TCT CCA* TCC TCT TCA CT (SEQ ID NO:6) IX: Oligomer CTG TCTCCA* TCC TCA* CT (SEQ ID NO:7) X:

where A* represents a nucleotide functionalized with acarbamate-2′-aminolinker moiety. Oligomers IX and X are antisensecompounds to the E2 region of the bovine papilloma virus-1 (BPV-1). Theoligonucleotides were synthesized on either a 10 μmol scale in the“Trityl-On” mode. Standard deprotection conditions (30% NH₄OH, 55° C.,24 hours) were employed. The oligonucleotides were purified by reversephase HPLC (Waters Delta-Pak C₄ 15 μm, 300 A, 25×100 mm column equippedwith a guard column of the same material). They were detritylated andfurther purified by size exclusion using a Sephadex G-25 column.

EXAMPLE 35 Conjugation of Oligonucleotides at the 2′-Position:Functionalization with Biotin

1. Single Site Modification

About 10 O.D. units (A₂₆₀) of Oligomer IX (see Example 34; approximately60 nmols based on the calculated extinction coefficient of 1.6756×10⁵),was dried in a microfuge tube. The oligonucleotide was dissolved in 200μl of 0.2M NaHCO₃ buffer and D-biotin-N-hydroxysuccinimide ester (2.5mg, 7.3 μmols) (Sigma, St. Louis, Mo.) was added followed by 40 μl DMF.The solution was let stand overnight. The solution was applied to aSephadex G-25 column (0.7×15 cm) and the oligonucleotide fractions werecombined. Analytical HPLC shoed nearly 85% conversion to the product.The product was purified by HPLC (Waters 600E with 991 detector,Hamilton PRP-1 column 0.7×15 cm; solvent A: 50 mM TEAA pH 7.0; B: 45 mMTEAA with 80% acetonitrile: 1.5 ml flow rate: Gradient: 5% B for first 5mins., linear (1%) increase in B every minute thereafter) and furtherdesalted on Sephadex G-25 to give the oligonucleotide conjugate:

Oligomer XI: CTG TCT CCA* TCC TCT TCA CT

wherein A* represents a nucleotide functionalized to incorporate abiotin functionality linked via a 2′-carbamate amino linking group atthe 2′-position of the designated nucleotide.

2. Multiple Site Modification

About 10 O.D. units (A₂₆₀) of oligomer X (see Example 34, approximately60 nmols) was treated utilizing the method of Example 8 withD-biotin-N-hydroxysuccinimide ester (5 mg) in 300 μl of 0.2M NaHCO₃buffer/50 μl DMF. Analytical HPLC showed 65% of double labeledoligonucleotide product and 30% of single labeled products (from the twoavailable reactive sites. HPLC and Sephadex G-25 purification gave theoligonucletide:

Oligomer XII: CTG TCT CCA* TCC TCT TCA* CT

wherein A* represents nucleotides functionalized to incorporate a biotinfunctionality linked via a 2′-carbamate aminolinker group to the2′-position of the designated nucleotide.

EXAMPLE 36 Assay Reagents Synthesis

1. Synthesis of Oligonucleotide-disuccinimidyl Suberate (DSS) Conjugate

An aliquot (10 O.D. units, 60 nmols) of Oligomer IX (Example 34) isevaporated to dryness and is dissolved in freshly prepared 0.1MNaHCO₃/50 nM EDTA (100 μl, pH 8.25). The solution is then treated with asolution of DSS (Pierce Chemical Co., Rockford, Ill.) (2.6 mg, 7 μmol)in 200 μl DMSO. The solution is stored at room temperature for 15minutes and then immediately applied to a Sephadex G-25 column (1×40 cm)that is previously packed and washed with water at 4° C. Theoligonucleotide fractions are combined immediately in a 25 mlpear-shaped flask and are rapidly frozen in dry ice/isopropyl alcoholand lyophilized to a powder.

2. Synthesis of oligonucleotide-protein (Alkaline Phosphatase) Conjugate

A solution of calf intestinal alkaline phosphatase (Boehringer Mannheim)(20.6 mg, 2.06 ml, 147 nmol) is spun at 4° C. in a Centriconmicroconcentrator at 6000 rpm until the volume is less than 50 μl . Itis then redissolved in 1 ml of cold Tris buffer (pH 8.5, 0.1M containing0.1 NaCl and 0.05M MgCl₂) and concentrated twice more. Finally, theconcentrate is dissolved in 400 μl of the same buffer. This solution isadded to the activated oligonucleotide from Previous Example and thesolution is stored at room temp. The product is diluted to approximately30 ml and applied to a Sephadex G-25 column (1×20 cm, chloride form)maintained at 4° C. The column is eluted with 50 nM Tris-Cl pH 8.5 untilthe UV absorbance of the fractions eluted reach near zero values. Thecolumn is then eluted with a NaCl salt gradient 0.05 M to 0.75 M (150 mleach). The different peaks are assayed for both oligonucleotide andalkaline phosphatase activity and the product bearing fractions arecombined. Typically the first peak will be excess enzyme, the secondpeak the oligonucleotide-protein conjugate and the third peak unreactiedoligonucletide. Isolation of the product from the product-bearingfractions via HPLC and desalting on Sephadex G-25 will yield anoligonucleotide of the sequence:

Oligomer XIII: CTG TCT CCA* TCC TCT TCA CT

wherein A* represents a nucleotide functionalized to incorporate analkaline phosphatase functionality linked via a2′-carbamate-aminolinker-sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate) linking group to the2′-position of the designated nucleotide.

EXAMPLE 37 Nuclease Protein-conjugated Oligonucleotides

As in Example 36, Oligomer IX is reacted with DSS reagent. The isolatedoligonucleotide-disuccinimidyl suberate conjugate is then furtherreacted with a lysine containing Nuclease RNase H using the method ofExample 36. This will give an oligonucleotide of the structure:

Oligomer CCC AGG CUC AGA*-3′-protein (SEQ ID NO:8) XIII:

wherein protein represents RNase H.

EXAMPLE 38 Internally Protein-conjugated 2′-Derivated Oligonucleotides

Utilizing the method of Example 36 the amino linker oligonucleotide ofExample 34 (Oligomer IX) is reacted with DSS reagent. The isolatedoligonucleotide-disuccinimidyl suberate conjugate is then furtherreacted with a lysine containing Staphylococcal Nuclease using themethod of Example 36. This will give an oligonucleotide of thestructure:

Oligomer XIV: CCC A*GG CUC AGA

wherein protein represents Staphylococcal Nuclease, the subscript “s”represents a phosphorothioate inter-nucleotide backbone linkage.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 8 <210> SEQ ID NO 1 <211> LENGTH: 10<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Se #quence<400> SEQUENCE: 1 tttttttttu                 #                  #                   #        10 <210> SEQ ID NO 2 <211> LENGTH: 10<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Se #quence<400> SEQUENCE: 2 tttttttttt                 #                  #                   #        10 <210> SEQ ID NO 3 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Se #quence<400> SEQUENCE: 3 tgcatccccc aggccacccu             #                  #                   # 20 <210> SEQ ID NO 4 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Se #quence<400> SEQUENCE: 4 tgcatccccc aggccaccat             #                  #                   # 20 <210> SEQ ID NO 5 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Se #quence<400> SEQUENCE: 5 tcgcatcgac ccgcccacta             #                  #                   # 20 <210> SEQ ID NO 6 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Se #quence<400> SEQUENCE: 6 ctgtctccat cctcttcact             #                  #                   # 20 <210> SEQ ID NO 7 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Se #quence<400> SEQUENCE: 7 ctgtctccat cctcact              #                  #                   #   17 <210> SEQ ID NO 8 <211> LENGTH: 12<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Se #quence<400> SEQUENCE: 8 cccaggcuca ga               #                  #                   #       12

What is claimed is:
 1. A method for detecting the presence or absence ofan RNA in a biological sample suspected of containing said RNAcomprising contacting said sample with a compound comprising a pluralityof linked nucleosides, wherein: each nucleoside includes a ribofuranosylsugar portion and a base portion; and at least one of said nucleosidesbears at a 2′—O—position or a 3′—O—position a substituent havingformula: —R_(A)—N—C(X)—O—R_(1a) or —C(X)—N(R_(1b))(R_(1c))  where: R_(A)is alkyl having from 1 to about 10 carbon atoms; R_(1a) is alkenylhaving 2 to about 10 carbon atoms; R_(1b) and R_(1c), independently, areH, R₂, R_(A), an amine protecting group or have formulaR_(A)—N(R_(1d))(R_(1e)), C(X)—R₂, C(X)—R_(A)-R₂, C(X)—Q—R_(A)-R₂, orC(X)—Q—R₂; R_(1d) and R_(1e), independently, are H, R₂, R_(A), an amineprotecting group or have formula C(X)—R₂, C(X)—R_(A)-R₂,C(X)-Q-R_(A)-R₂, or C(X)—Q—R₂; R₂ is a steroid molecule, a reportermolecule, a lipophilic molecule, a reporter enzyme, a peptide, aprotein, includes folic acid, or has formula —Q—(CH₂CH₂—Q—)_(x)—R₃; X isO or S; each Q is, independently, is NH, O, or S; x is 1 to about 200;R₃ is H, R_(A), C(O)OH, C(O)OR_(A), C(O)R₄, R_(A)—N₃, or R_(A)—NH₂; R₄is Cl, Br, I, SO2R5 or has structure:

m is 2 to 7; and R₅ alkyl having 1 to about 10 carbon atoms.
 2. A methodfor detecting the presence or absence of an RNA in a biological samplesuspected of containing said RNA comprising contacting said sample witha compound comprising a plurality of linked nucleosides, wherein: eachnucleoside includes a ribofuranosyl sugar portion and a base portion;and at least one of said nucleosides includes a pyrimidine base whichbears at its 5-position a substituent having formula:—R_(A)—O—C(X)—N(R_(1b))(R_(1c))  where: R_(A) is alkyl having from 1 toabout 10 carbon atoms; R_(1b) and R_(1c), independently, are H, R₂,R_(A), an amine protecting group or have formulaR_(A)—N(R_(1d))(R_(1e)), C(X)—R₂, C(X)—R_(A)-R₂, C(X)—Q—R_(A)-R₂, orC(X)—Q—R₂; R_(1d) and R_(1e), independently, are H, R₂, R_(A), an amineprotecting group or have formula C(X)—R₂, C(X)—R_(A)-R₂,C(X)—Q—R_(A)-R₂, or C(X)—Q—R₂; R2 is a steroid molecule, a reportermolecule, a lipophilic molecule, a reporter enzyme, a peptide, aprotein, includes folic acid, or has formula —Q—(CH₂CH₂—Q—)_(x)—R₃; X isO or S; each Q is, independently, is NH, O, or S; x is 1 to about 200;R₃ is H, R_(A), C(O)OH, C(O)OR_(A), C(O)R₄, RA—N₃, or R_(A)—NH₂; R₄ isCl, Br, I, SO₂R₅ or has structure:

m is 2 to 7; and R₅ alkyl having 1 to about 10 carbon atoms.